Pure bone marrow fibroblasts, macrophages and endothelial cells were cultured in Iscove-modified Dulbecco's medium. RT—PCR was used to determine the expression of inhibitory cytokine mRNAs in these cell types. Serum-free conditioned medium was collected from each cell type and ultrafiltration was performed with a centriprep 10. The retentate contained substances whose molecular weights were >10 kD, whilst the filtrate contained substances with molecular weights <10 kD. The effect of conditioned media and their components on colony forming unit-granulocyte-macrophage (CFU-GM) were investigated.
The results showed: (1) six cytokines, MIP-1α, MIP-2, TGF-β, TNF-α, IFN-γ and Tβ4, inhibited the growth of CFU-GM when murine WEHI-3 conditioned medium was added to the culture system as a source of colony stimulation. (2) The original endothelial cell conditioned medium (E-CM) did not affect the production of CFU-GM, but the >10 kD component of E-CM increased its production, and the <10 kD component decreased it. Both fibroblast conditioned medium (F-CM) and the >10 kD component of F-CM stimulated proliferation of CFU-GM, but the <10 kD component suppressed it. All three components of macrophage conditioned medium (M-CM) inhibited the growth of CFU-GM. (3) Expression of four of the mRNAs, namely MIP-2, TNF-α, INF-γ and Tβ4, was seen in all three types of stromal cells, while TGF-β mRNA was only seen in endothelial cells and macrophages, and MIP-1α mRNA in endothelial cells and fibroblasts. The inhibitors TGF-β, MIP-1α, and Tβ4have an inhibitory effect on the growth of CFU-GM, but TNF-α, INF-γ and MIP-2 do not.